1. Technical Field
The subject invention relates to a method of producing and purifying large quantities of biosynthetic proteins.
The gene which codes for a retroviral protease is placed between the binding domain of a gene which codes for a binding protein and a gene coding for the target protein of interest. The fused gene construct is inserted in an expression vector which is then introduced into a host cell.
2. Background Information
Gene fusions have been used successfully to express and purify proteins produced from recombinant DNA molecules. A common class of fusions involves .beta.-galactosidase whereby the fusion protein is expressed in E. coli using the ribosome binding site and the translational start of lacZ (Shuman et al., J. Biol. Chem. 255:168-74 (1980) and Silhavy et al., "Experiments With Gene Fusions," (Cold Spring Harbor Laboratory 1984)). Such hybrid proteins can be purified by affinity chromatography using anti-.beta.-galactosidase affinity columns. A similar fusion system uses protein A from Staphylococcus aureus (Lowenadler et al., EMBO 5:2393-98 (1986) and Valerie et al., Gene 58:99-107 (1987)) or a synthetic IgG-binding domain of protein A to purify fusion proteins by binding to an IgG resin (Lowenadler et al., Gene 58:87-97 (1987)).
The most widely used enzyme for isolating the target protein from the fusion protein involves a protease cleavage site specific to factor Xa protease. Employing the factor Xa protease recognition site to release the target protein, in the present studies, was not successful even by providing an Arg-Gly bond. The entire factor Xa gene has never, in the past, been inserted between the binding protein and the target protein.
Most serine proteases either fail to cleave Arg-Pro bonds or do so very slowly. Factor Xa has a strong preference for Arg-Gly bonds in addition to its specificity for the Ile-Glu-Gly-Arg sequence. The cleavage is dependent on the nature of target protein and the conformation of the fusion protein, in addition to several other factors.
A protease of recent interest is the one produced by the human immunodeficiency virus. This virus is the causative agent of the acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi et al., Science 220:868-70 (1983) and Gallo et al., Science 224:500-502 (1984)). The genome size mRNA, like that of other retroviruses, has a gag-pol-env organization and is translated into the Pr55gag and Pr160gag pol polyproteins. HIV-1 like all other replication competent retroviruses, encodes a protease (PR) which is responsible for the specific cleavage of Pr55gag and Pr160gag pol into the mature gag derived structural proteins p17, p24, p7 and p6, and the pol derived protease (PR), reverse transcriptase (RT) and the endonuclease, the integration protein (IN). Protease deficient mutants of the murine leukemia virus and of HIV were shown to produce immature noninfectious virus particles (see Meek et al., Nature 343:90-92 (1990) and McQuade et al., Science 247:454-56 (1990)). These studies suggested that the protease may be a potential target for drug therapy (see also Moore et al., Biochemical and Biophysical Research Communications 159:420-25 (1989)). The requirements for sufficient quantities of enzyme for structural and biochemical studies necessitated expression of the protease in E. coli and yeast. (Kraussich et al., In Annual Review of Biochemistry (Richardson et al., eds.) Vol. 55, pp. 701-54 (1984) and Oroszlan et al., In Current Topics in Microbiology and Immunology (Swanstrom et al., eds. (1990)). Structural analysis of the active human immunodeficiency virus-1 (HIV-1) protease has shown that it is a homodimer possessing one active site. The active site of the protease contains two sequences of AspThrGly, common with the other known aspartic proteases (Wlodawer et al., Science 245:616-21 (1989) and Lapatto et al., Nature 342:299-302 (1989)). Recently, it was demonstrated that inhibitors of the HIV-1 protease arrest the maturation of HIV-1-like particles (Heek et al., Nature 343:90-92 (1990) and McQuade et al., Science 247:454-56 (1990)). Expression of the protease gene (297 bp) in E. coli provided low recovery of the pure protease (McKeever et al., J. Bio. Chem. 264:1919-21 (1989)). The protease gene expressed as a fusion with flanking truncated gag-pol region sequences resulted in the self-processing of the protease in E. coli (Debouk et al., Proc. Natl. Acad. Sci. USA 84:8903-06 (1987)).
Furthermore, a study was done on the expression of the HIV-1 protease as a fusion with the LacZ of E. coli (Giam et al., J. Biol. Chem. 263:14617-20 (1988)). In this case, the expressed fusion was partially soluble, and the majority of the product was found in the inclusion bodies. Consequently, a single column purification of the fusion product, immediately after lysis of the cells, cannot be performed since the fusion protein is insoluble. Purification of the fusion protein from the inclusion bodies involves considerable loss in the final amount of the protease recovered.
In another system, the target protein was fused to the maltose binding protein (MBP) coded for by the malE gene of E. coli (Amann et al., Gene 67:21-30 (1985) and Maina et al., Gene 74:365-73 (1988)).
This latter approach uses a crosslinked amylose resin as an affinity matrix to purify the MBP fusion protein. A high yield of pure fusion protein is obtained using a single purification step carried out under non-denaturing conditions. This system of expression would allow for the separation of the target domain from the MBP domain by the site-specific proteolytic cleavage (factor Xa, specific cleavage at the tetrapeptide, IleGluGlyArg) after purification. Thus, if the recognition site is placed before the target protein domain, factor Xa will cleave specifically at the site releasing the target protein without any additional N-terminal residues.
Unfortunately, there are many disadvantages to this procedure. For example, as the present inventors observed, utilizing clone A, the target protein does not undergo cleavage using the factor 10a protease.
Furthermore, there are many advantages to the use of the HIV-1 protease of the present invention rather than the factor Xa protease. Although literature exists which shows that the factor Xa protease can cleave, in some cases, fusion proteins after denaturation, it has never been incorporated as one of the components in the fusion protein, like the HIV-1 protease. The HIV-1 protease, as one of the components of the fusion protein of the present invention, has been shown to renature and cleave itself completely from the fusion protein. Furthermore, the HIV-1 protease, being a smaller enzyme than the factor Xa protease, would be expected to more readily effect cleavage at the appropriate cleavage site in the fusion protein and thereby more efficiently release the target protein from the fusion protein.